体外HBV感染的人肝癌Huh7细胞系中葡萄糖神经酰胺合成酶活性的变化

Change in the activity of glucosylceramide synthase in human hepatoma cell line with hepatitis B virus infection in vitro

目的探索糖鞘脂代谢合成关键酶葡萄糖神经酰胺(GCS)在体外HBV感染Huh7细胞中的活性变化。方法收集2019年6月—8月就诊于兰州大学第一医院感染科的9例初治急性乙型肝炎患者的血液标本,另选7例健康体检者的血液标本作为对照。利用HBV感染者血清中高拷贝HBV颗粒(>9.9×10^(7) IU/ml)接种Huh7细胞系(感染组),对照组用健康体检者的血清共培养。检测HBV感染细胞胞浆中HBsAg和HBV DNA的表达量以及GCS的活性变化与病毒的相关性。计量资料多组间数据比较采用单因素方差分析,进一步两两比较采用LSD-t检验。相关性分析采用Pearson检验。结果感染组细胞较同期对照组细胞数量明显减少、细胞体积肿胀和细胞膜破碎。感染组:感染4 h、8 h、2 d和5 d后胞浆中HBsAg表达量均高于感染前(P<0.05);与感染4 h(16.67±11.55)IU/ml相比,感染8 h(112.01±25.94)IU/ml、2 d(328.01±103.50)IU/ml和5 d(101.60±49.84)IU/ml后胞浆中HBV DNA表达量呈明显升高趋势(P值均<0.001),并在感染2 d时HBV DNA表达量达到最高。在HBV感染期间,随着病毒复制的增加,GCS活性呈现出增高的特点,从感染4 h(126.21±9.59)IU/ml开始逐渐升高,并于感染2 d时(226.53±36.27)IU/ml达到高峰,感染2 d时与对照组(136.50±15.44)IU/ml比较,差异有统计学意义(t=3.956,P=0.0167)。GCS活性与HBV DNA水平存在显著正相关(r=0.5768,P=0.0471)。结论HBV感染者血清中高拷贝HBV颗粒能成功感染Huh7细胞系,并在一定程度上模拟了HBV感染的体外特点。GCS活性可能与HBV的感染有关,表明糖鞘脂合成代谢与HBV可能存在密切的联系。

Objective To investigate the change in the activity of glucosylceramide synthase,the key enzyme in glycosphingolipid metabolism and synthesis,in Huh7 cells infected by hepatitis B virus(HBV)in vitro.Methods Blood samples were collected from nine previously untreated patients with acute hepatitis B who attended Department of Infectious Diseases,The First Hospital of Lanzhou University,from June to August,2019,and the blood samples collected from seven healthy individuals who underwent physical examination were established as control.Huh7 cells were inoculated with the high-copy HBV particles(>9.9×10^(7) IU/ml)in the serum of patients with HBV infection(infection group),and Huh7 cells co-cultured with the serum of healthy individuals were established as control group.The expression levels of HBsAg and HBV DNA in the cytoplasm of HBV-infected Huh7 cells were measured,and the correlation between GCS activity and virus was analyzed.A one-way analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups,and a Pearson correlation analysis was performed.Results Compared with the control group,the infection group had a significant reduction in the number of cells,an increase in cell volume,and cell membrane fragmentation.The infection group had a significant increase in the expression of HBsAg in cytoplasm at 4 hours,8 hours,2 days,and 5 days after infection(P<0.05);the expression level of HBV DNA tended to increase significantly from 4 hours after infection to 8 hours,2 days,and 5 days after infection(16.67±11.55 IU/ml vs 112.01±25.94 IU/ml/328.01±103.50 IU/ml/101.60±49.84 IU/ml,P<0.001),with the highest level at 2 days after infection.During HBV infection,the activity of GCS gradually increased with the increase in viral replication from 4 hours after infection(126.21±9.59 IU/ml)and reached a peak at 2 days after infection(226.53±36.27 IU/ml),with a significant difference between the infection group and the control group at 2 days after infection(226.53±36.27 IU/ml vs 136.50±15.44 IU/ml,t=3.956,P=0.0167).The activity of GCS was positively correlated with HBV DNA level(r=0.5768,P=0.0471).Conclusion Huh7 cells are successfully infected with the high-copy HBV particles in the serum of patients with HBV infection,which mimics the characteristics of HBV infection in vitro to a certain degree.The activity of GCS may be associated with HBV infection,suggesting that glycosphingolipid synthesis and metabolism may be closely associated with HBV.